There are two isoforms of the nonmuscle myosin heavy chain, MHC-A and MHC-B, that are encoded by two separate genes. Most tissues express different relative ratios of MHC-A and MHC-B with the extremes being platelets, which express only MHC-A, and brain tissue which is enriched in MHC-B. Although previous work has suggested a unique role for MHC-B based on its phosphorylation by p34-cdc2 kinase during meiosis (Kelley et al., J. Biol. Chem. 270, 1395, 1995), to date, the specific functions of these isoforms have not been identified. We compared the enzymatic activities as well as the subcellular localizations of these isoforms using the cultured Xenopus kidney epithelial cell line, A6. MHC-A and MHC-B were purified from A6 cells by immunoprecipitation with isoform- specific peptide antibodies followed by elution with their cognate peptides. Using an in vitro motility assay, we found that the velocity of movement of actin filaments by MHC-A (V = 0.725+0.166) was four-fold faster than that by MHC-B (0.187+.032). Likewise, the Vmax of the actin-activated Mg2+-ATPase activity of MHC-A was two-fold greater than that of MHC-B (0.187+.032). Immunofluorescence microscopy studies using isoform-specific antibodies demonstrated distinct localizations for MHC- A and MHC-B in interphase cells. MHC-B was present in the cell cortex and diffusely arranged in the cytoplasm. In contrast, MHC-A was notably absent from the cell periphery and was arranged in a fibrillar type staining pattern in the cytoplasm. Double staining for F-actin with rhodamine-phalloidin or microtubules with anti-tubulin antibodies as well as results from treatment of cells with nocodazole or cytochalasin- B showed that both MHC-A and MHC-B localize with F-actin, but not microtubules. The observed localizations of MHC-A and MHC-B together with data demonstrating that these myosins have markedly different enzymatic activities strongly suggests that MHC-B is involved in dynamic aspects at the cell periphery, such as protrusion, while MHC-A has a more structural role in cell adhesion.